Germline and somatic mutations in the Apc (Adenomatous polyposis coli ) gene are thought to be seminal genetic events in the etiology of human and murine colorectal cancer. ApcMin mice carry a germline mutation in the Apc gene and experience reduced lifespan due to adenocarcinoma burden. Wild type, but not mutated, APC binds to and regulates b-catenin, the mammalian homolog of armadillo required for cadherin-mediated cell adhesion. b-catenin released from its binding to membrane E-cadherin forms a heterodimer with Tcf/LEF and functions as a transcriptional factor. To study the regulation of this pathway, we used two conditional immortal murine intestinal epithelial cell lines contrasting in Apc genotype ("Immortomouse"/Min Colonic Epithelia (IMCE), Apc +/-; Young Adult Mouse Colon epithelia (YAMC), Apc +/+). We have demonstrated that IMCE cells which have defective degradation of b-catenin, have higher levels of b-catenin/LEF-1 transcriptional factor by EMSA and higher expression of COX-2 than YAMC cells in response to lipopolysaccharide (LPS) and interferon-g (IFN-g). The critical role of nitric oxide (NO) in this response was shown by the abrogation of the LPS, IFN-g effect by inhibitors of nitric oxide synthase II. Additionally, NO donors increased b-catenin/LEF-1 formation by EMSA as well as the expression of COX-2. That the effect was mediated by the availability of b-catenin was supported by the differential response in IMCE and YAMC cells and by the direct demonstration of free, cytoplasmic b-catenin in response to NO treatment. Our current work is focused on the mechanism by which NO increases free, unbound b-catenin. Preliminary findings suggest that NO stimulates the degradation of membrane bound E-cadherin with the concomitant release of b-catenin from the cytoplasmic E-cadherin binding site. Using an antibody recognizing the extracellular domain of E-cadherin, we found that treatment with NO donors markedly increased E-cadherin degradation products accumulating in the medium. Since metalloproteinases mediate the degradation of E-cadherin, we tested several inhibitors of metallo-proteinases and found that they not only blocked the effect of NO on E-cadherin degradation but also abrogated its effect on the formation of b-catenin/LEF-1 transcriptional complexes. These findings suggest that the activation of metalloproteinases by NO releases free b-catenin from E-cadherin to form b-catenin/LEF-1 transcriptional complexes. Using both synthetic metalloproteinase inhibitors and tissue inhibitors of metalloproteinases (TIMPs), we are identifying the specific metalloproteinase activated by NO and characterizing the mechanism of this activation. The findings using TIMPs also suggest that NO may regulate transcriptional factors other than b-catenin/LEF-1. The effects of NO on several transcriptional factors are pursued using promoter-reporter gene constructs and co-transfections with expression constructs for several transcriptional factors. We have discovered synergistic stimulation between NO and several transcriptional factors and the mechanism for this stimulation has become an important focus of this project.